Kinetic analyses of divalent cation-dependent EcoRV digestions on a DNA-immobilized quartz crystal microbalance
Kinetic analyses of divalent cation-dependent EcoRV digestions on a DNA-immobilized quartz crystal microbalance
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DOI：
10.1016/j.ab.2006.11.030
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发表时间：
2007-02-15
影响因子：
2.9
通讯作者：
Okahata, Yoshio
中科院分区：
文献类型：
--
作者：
Takahashi, Shuntaro;Matsuno, Hisao;Okahata, Yoshio
在DNA固定的27 MHz石英晶体微天平(QCM)上，研究了IIP限制性内切酶EcoRV的酶消化作用。实时观察EcoRV的酶结合过程和DNA裂解过程之后，QCM上的频率(质量)发生变化，这取决于二价阳离子，如钙或镁。在有Ca~(2+)存在的情况下，EcoRV可与DNA发生特异性结合，而不需要催化过程。另一方面，在Mg2+存在下，从QCM频率的变化中可以连续观察到酶与特定DNA的结合(质量增加)和位点特异性切割反应(质量减少)。根据频率(质量)变化的时间历程，可以确定每个动力学参数，即结合速率常数(k(On))、解离速率常数(k(Off))、EcoRV与DNA的解离常数(K-d)和裂解反应的催化速率常数(k(Cat))。EcoRV在钙离子存在下的结合动力学参数与在镁离子存在下的结合过程和切割过程的动力学参数是一致的。QCM法测得的kcat值与其他方法测得的kcat值基本一致。这项研究是首次在一台设备上同时测定IIP限制性内切酶的k(开)、k(关)和k(CAT)。(C)2006 Elsevier Inc.保留所有权利。
Enzymatic digestion with a type IIP restriction endonuclease EcoRV was investigated on a DNA-immobilized 27-MHz quartz crystal microbalance (QCM). Real-time observations of both the enzyme binding process and the DNA cleavage process of EcoRV were followed by frequency (mass) changes on the QCM, which were dependent on divalent cations such as Ca2+ or Mg2+. In the presence of Ca2+, the site-specific binding of EcoRV to DNA could be observed, without the catalytic process. On the other hand, in the presence of Mg2+, both the binding of the enzyme to the specific DNA (mass increase) and the site-specific cleavage reaction (mass decrease) could be observed continuously from QCM frequency changes. From time courses of frequency (mass) changes, each kinetic parameter, namely binding rate constants (k(on)), dissociation rate constants (k(off)), dissociation constants (K-d) of EcoRV to DNA, and catalytic rate constant (k(cat)) of the cleavage reaction, could be determined. The binding kinetic parameters of EcoRV in the presence of Ca2+ were consistent with those of the binding process followed by the cleavage process in the presence of Mg2+. The kcat value obtained by the QCM method was also consistent with that obtained by other methods. This study is the first to simultaneously determine k(on), k(off), and k(cat) for a type IIP restriction endonuclease on one device. (c) 2006 Elsevier Inc. All rights reserved.